U.S. patent number 4,745,161 [Application Number 06/846,707] was granted by the patent office on 1988-05-17 for soluble and biodegradable polyamino acid activated for bonding of biologically active compound.
This patent grant is currently assigned to Ceskoslovenska akademie ved. Invention is credited to Jaroslav Drobnik, Frantisek Rypacek, Vladimir Saudek.
United States Patent |
4,745,161 |
Saudek , et al. |
May 17, 1988 |
Soluble and biodegradable polyamino acid activated for bonding of
biologically active compound
Abstract
The invention pertains to copolymers activated for bonding of
biologically active compounds and to a method for preparation
thereof. Soluble and biodegradable copolymers activated for bonding
of biologically active compounds which have their main chain formed
from units of aspartic acid or glutamic acid, or both, or also from
units of other amino acids, according to the general formula I
##STR1## where n is 1 or 2, m is 1 or 2, a+b+c is 100, a is 0.5 to
100, b is 0 to 99.5, c is 0 to 80; the substituent R.sup.1 is
--NH--NH.sub.2, --N.tbd.N or ##STR2## where x is 2 to 6, R.sup.2 is
--NH(CH.sub.2).sub.x OH where x=2 to 6; and R.sup.3 is methyl,
2-propyl, 2-methylpropyl, butyl, 2-butyl, benzyl or
4-hydroxybenzyl, and the molecular weight is 5,000 to
1,000,000.
Inventors: |
Saudek; Vladimir (Prague,
CS), Rypacek; Frantisek (Prague, CS),
Drobnik; Jaroslav (Prague, CS) |
Assignee: |
Ceskoslovenska akademie ved
(CS)
|
Family
ID: |
5364033 |
Appl.
No.: |
06/846,707 |
Filed: |
April 1, 1986 |
Foreign Application Priority Data
|
|
|
|
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Apr 10, 1985 [CS] |
|
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2634/85 |
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Current U.S.
Class: |
525/420; 528/322;
528/310; 528/328 |
Current CPC
Class: |
C08G
69/10 (20130101); A61K 47/64 (20170801); C08G
73/1092 (20130101) |
Current International
Class: |
A61K
47/48 (20060101); C08G 73/00 (20060101); C08G
69/00 (20060101); C08G 69/10 (20060101); C08G
73/10 (20060101); C08G 069/48 () |
Field of
Search: |
;525/420
;528/310,328 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
|
17084 |
|
Aug 1964 |
|
JP |
|
8590 |
|
Jan 1974 |
|
JP |
|
Other References
Cols. 1-2, 35-36, U.S. Pat. No. 3,306,875, Hay..
|
Primary Examiner: Anderson; Harold D.
Attorney, Agent or Firm: Leydig, Voit & Mayer
Claims
What is claimed is:
1. A biodegradable copolymer activated for bonding of biologically
active compounds, wherein the main chain of the copolymer is formed
from the units of aspartic acid or glutamic acid, or from both
these units, or also from units of other amino acids, according to
the general formula I ##STR7## where n is 1 or 2, m is 1 or 2,
a+b+c is 100, a is 0.5 to 100, b is 0 to 99.5, c is 0 to 80,
substituent R.sup.1 is --NH--NH.sub.2, --N.tbd.N or ##STR8## where
x is 2 to 6; R.sup.2 is --NH(CH.sub.2).sub.x OH where x is 2 to 6;
and R.sup.3 is methyl, 2-propyl, 2-methylpropyl, butyl, 2-butyl,
benzyl or 4-hydroxybenzyl.
2. Method for preparation of the copolymer according to claim 1,
wherein a polymer having units of the general formula II ##STR9##
where R is methyl or benzyl, n is 1 or 2, m is 1 or 2, a+b+c is
100, a is 0.5 to 100, b is 0 to 99.5, c is 0 to 80; R.sup.2 is R or
is the same as in claim 1; and R.sup.3 is the same as in the
general formula I; is treated at 0.degree. C. to 60.degree. C. with
a mixture containing primary or secondary amino alkylalcohol,
wherein the alkyl is a linear or branched hydrocarbon chain having
2 to 6 carbon atoms, and hydrazine.
Description
BACKGROUND
The invention pertains to soluble and biodegradable copolymers
which are activated for bonding of biologically active
compounds.
Biologically active compounds bonded on soluble synthetic polymers
have been proposed during recent years as suitable types of so
called "prodrugs" for medical applications. Such bonded compounds
enable, for example, longer excretion halftimes for active
components, affects at active sites in the body, combining several
activities, etc. Poly(aspartic acid) and poly(glutamic acid) and
their derivatives are prospective polymers for such applications.
Reactive functional groups must to be introduced into the polymers
to bond chosen compounds. Poly(succinimide) is usually used for the
synthesis, of poly(aspartic acid) as the reactive intermediate
which is allowed to react with a reactive amine carrying a suitable
functional group. However, this procedure always affords the
racemic polymer which, moreover, contains both .alpha. and .beta.
peptide bonds in the main chain and, consequently, the resulting
polymer is biodegradable with great difficulty.
The biodegradable derivatives of activated poly(aspartic acid) may
be obtained by the polymerization of N-carboxyanhydrides of the
.beta.-monoester of the pertinent amino acid by deprotection of the
carboxylic group through ester cleavage, and the subsequent
activation of carboxylic group, e.g. with carbodiimide or formic
esters. A disadvantage of this procedure is the complicated
multistep synthesis, low yield, possible accumulation of
undesirable side reactions, and incomplete conversion in the
individual steps. In addition to this, polyanions with free
carboxylic groups are not very suitable for practical medical
application, and it is more convenient to prepare non-ionogenic
derivatives with hydroxyl groups in the side chain. This requires,
indeed, further reaction with aminoalcohols after bonding of a
biologically active compound.
Preparation of poly(N-hydroxyethylglutamine) carrying
1-.beta.-D-arabinofuranosylcytosine (ara-C) in the side chain may
serve as an example of this procedure: Poly(.gamma.-benzyl
glutamate) was transferred to poly(glutamic acid) and ara-C and
then aminoethanol were incorporated by means of isobutyrylcarbonyl
chloride (Kato et al., Cancer Res. 44, 25 (1984)). The copolymer of
glutamic acid and glutamic acid hydrazide was also prepared from
poly(glutamic acid) by the reaction with tert-butyl carbazate by
means of N-ethyloxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The
tert-butyl group was split off after reaction. This copolymer was
employed for bonding of adriamycin by means of an acylhydrazone
bond (Heeswijk et al., Rec. Adv. Drug Delivery Systems, Anderson
Kim ed., Plenum Publishing Corporation, 1984, p. 77-100).
If the copolymer of hydroxyalkylglutamine and glutamic acid is
prepared from polyalkylglutamate by an incomplete aminolysis of the
ester groups followed by the hydrolysis, the carboxylic groups thus
formed cannot be simply activated for the reaction with a
biologically active compound without the parallel undesirable
reaction between the hydroxyl groups of hydroxyalkylglutamine and
carboxylic groups.
DISCLOSURE OF THE INVENTION
It has been found now that aminoalcohols catalyze the
hydrazinolysis of esters so that it is possible to prepare
copolymers of hydroxyalkylglutamines and hydroxyalkyl aspartagines
with the corresponding hydrazides of monomeric units of glutamic
and aspartic acids very easily by a single-step reaction. The
starting material are polymeric esters, for example,
poly(.gamma.-benzyl glutamate) or poly(.gamma.-methyl glutamate) or
poly(.beta.-alkyl aspartates), their mutual copolymers or
copolymers with other amino acids, which are treated with a mixture
of aminoalcohol and hydrazine. The ratio of these components in the
reaction mixture controls the ratio of hydroxyalkylamide and
hydrazide units in the resulting product. The reaction may be
further accelerated by addition of 2-hydroxypyridine.
The copolymers with hydrazides of aspartic acid may also be
prepared from poly(succinimide) or from copolymers of succinimide
with other amino acids, again by the simultaneous reaction of
aminoalcohol and hydrazine.
According to the invention, it is possible to bind compounds with
aldehyde or ketone groups directly to the hydrazide-containing
copolymers. Another way is the oxidation of hydrazide to azide
which then easily reacts with both aromatic and aliphatic amines.
The reaction may be carried out either in water or in organic
solvents. The invention enables, in this way, the direct bonding of
chosen compounds with an amino group, or the preparation of
intermediates by bonding amines of the type ##STR3## where n=0 to
6, which are then activated by diazotization.
The object of this invention is a soluble and biodegradable
copolymer, activated for the bonding of biologically active
compounds, wherein the main chain is formed from the units of
aspartic acid or glutamic acid, or from both these units, or also
from units of other amino acids, according to the general formula I
##STR4## where n is 1 or 2, m is 1 or 2, a+b+c is 100, a is 0.5 to
100, b is 0 to 99.5, c is 0 to 80; the substituent R.sup.1 is
--NH--NH.sub.2 --, --N.tbd.N or ##STR5## where x is 2 to 6; R.sup.2
is --NH(CH.sub.2).sub.x OH where x is 2 to 6; and R.sup.3 is
methyl, 2-propyl, 3-methylpropyl, butyl, 2-butyl, benzyl or
4-hydroxybenzyl, and the molecular weight is 5,000 to
1,000,000.
The object of this invention is further a method for preparation of
the copolymer wherein a polymer having units of the general formula
II ##STR6## where R is methyl or benzyl, n is 1 or 2, m is 1 or 2,
a+b+c is 100, a is 0.5 to 100, b is 0 to 99.5, c is 0 to 80,
R.sup.2 is R or is the same as in the general formula I, and
R.sup.3 is the same as in the general formula I, is treated at a
temperature of from 0.degree. C. to 60.degree. C. with a mixture
containing primary or secondary aminoalkylalcohol, wherein the
alkyl is a linear or branched hydrocarbon chain having 2 to 6
carbon atoms, and hydrazine.
The object of the invention is also a method for the preparation of
copolymers of aspartic acid, wherein poly(succinimide) reacts at
temperature 0.degree. C. to 60.degree. C. with a mixture containing
primary or secondary aminoalkylalcohol, wherein the alkyl is a
linear or branched hydrocarbon chain having 2 to 6 carbon atoms,
and hydrazine.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention is illustrated in the following examples, which,
however, do not limit the scope of the invention by any means.
EXAMPLE 1
Poly(.gamma.-benzyl L-glutamate) (2 g) was dissolved in 20 ml of
dimethylacetamide and then a mixture of 86 .mu.l of hydrazine and
1.5 ml of ethanolamine was dropwise added under vigorous stirring
at ambient temperature. The stirring was continued for 20 min, the
reaction mixture was then allowed to stand at 60.degree. C. for 4
days, and then poured into 200 ml of ethanol containing 5 ml of
acetic acid; the precipitate was separated by filtration, dissolved
in the acidified water, dialyzed against water, and lyophilized.
The product was 1.01 g of the copolymer of .gamma.-hydrazide of
L-glutamic acid (11 mol.%) and N.sup..gamma.
-(2-hydroxyethyl)-L-glutamine (89 mol.%).
EXAMPLE 2
Poly(succinimide) (2 g) was dissolved in 20 ml of dimethylformamide
and a mixture of 144 .mu.l of hydrazine and 2.5 ml of ethanolamine
was dropwise added under stirring and cooling to 0.degree. C. The
reaction mixture was then stirred at ambient temperature overnight
and worked out in the same way as in example 1. The product was 1.9
g of the copolymer of .alpha.,.beta.-hydrazide of D,L-aspartic acid
(12 mol.%) and N.sup..alpha.,.beta. -(2-hydroxyethyl)-D,L-aspartic
acid (88 mol.%).
EXAMPLE 3
Poly(.gamma.-benzyl L-glutamate) (1 g) was overcast with a mixture
of 2.5 ml ethanolamine and 14 .mu.l of hydrazine and allowed to
stand at 60.degree. C. for 4 days. The resulting solution was
precipitated with 50 ml of ethanol containing 4 ml of acetic acid,
the precipitate was washed with ethanol and ether and dried. The
product was 0.77 g of the analogous copolymer as in example 1 which
contained 1.3 mol.% of hydrazide.
EXAMPLE 4
The reaction was carried out in the same way as in example 1, but
2-hydroxybutylamine was used instead of ethanolamine. The product
was 1.0 g of the copolymer of .gamma.-hydrazide of L-glutamic acid
(10 mol.%) and N.sup..gamma. -(2-hydroxybutyl)-L-glutamine.
EXAMPLE 5
The reaction was carried out in the same way as in example 1, with
the distinction that 3-hydroxybutylamine was used instead of
ethanolamine. The product was 1.0 g of the copolymer of
.gamma.-hydrazide of L-glutamic acid (10 mol.%) and N.sup..gamma.
-(3-hydroxybutyl)-L-glutamine.
EXAMPLE 6
The copolymer of .gamma.-benzyl L-glutamate with 3 mol.% of
L-phenylalanine (1 g) was dissolved in 10 ml of dimethylacetamide
and then 0.75 ml of the solution containing 3.1 mol.% of hydrazine;
and 10 mol.% of hydroxypyridine in ethanolamine was dropwise added
under vigorous stirring at ambient temperature. The preparation was
continued as in example 1. The product was the terpolymer of
.gamma.-hydrazide of glutamic acid (9.6 mol.%), phenylalanine (3
mol.%) and N.sup..gamma. -(2-hydroxyethyl)-L-glutamine.
EXAMPLE 7
The reaction was carried out in the same way as in example 6, with
the distinction that the copolymer of .gamma.-benzyl L-glutamate
with 6 mol.% of leucine and the solution containing 2.5 mol.% of
hydrazine in ethanolamine were used. The product was 0.48 g of the
copolymer of .gamma.-hydrazide of glutamic acid (8.4 mol.%),
leucine (6 mol.%) and N.sup..gamma.
-(2-hydroxyethyl)-L-glutamine.
EXAMPLE 8
The reaction was carried out in the same way as in example 6, with
the distinction that the copolymer of .gamma.-benzyl L-glutamate
with 4 mol.% of valine and the solution of 1.9 mol.% hydrazine in
ethanolamine were used. The product was 0.51 g of the terpolymer of
.gamma.-hydrazide of glutamic acid (6.6 mol.%), valine (4 mol.%)
and N.sup..gamma. -(2-hydroxyethyl)-L-glutamine.
EXAMPLE 9
The polymer resulting in example 1 was dissolved in 3 ml of 0.1
mol/l HCl and 1 ml of 0.1 mol/l sodium nitrite (NaNO.sub.2) and,
after 5 min, the suspension of 39 mg of
N-(6-aminohexyl)-2,4-dinitroanilide (AHDN) dihydrochloride in 20 ml
of 0.2 mol/l sodium hydrogencarbonate (NaHCO.sub.3) were
successively added under stirring and cooling to 0.degree. C. After
2 hours of stirring, the solution was transferred into a dialysis
tube, dialyzed against water and freeze-dried. The resulting
preparation contains N-(6-aminohexyl)-2,4-dinitroanilide (AHDN)
bonded to 11 mol.% of monomer units.
EXAMPLE 10
To the solution of 100 mg of the polymer prepared in example 2 in 2
ml of dimethylsulfoxide (DMSO) it was successively added under
stirring and cooling to 0.degree. C. 1 ml of 0.1 mol/l hydrochloric
acid (HCl) in methanol, a mixture of 0.5 ml of butyl nitrite and
4.5 ml of dimethylsulfoxide (DMSO) and, after 5 min, a solution of
0.139 ml of triethylamine and 39 mg of
N-(6-aminohexyl)-2,4-dinitroanilide (AHDN) dihydrochloride in 10 ml
of dimethylsulfoxide (DMSO). After 2 h, the reaction mixture was
dialyzed against water and freeze-dried. The resulting preparation
contained N-(6-aminohexyl)-2,4-dinitroanilide bonded to 11% of
monomer units.
EXAMPLE 11
45 mg of the polymer prepared in example 6 was disolved in 10 ml of
0.1 mol/l hydrochloric acid and cooled to 0.degree. C. Under
stirring and cooling 10 ml of 0.1 mol/l sodium nitrite was added to
it followed, after 5 min by a solution of 10 mg of adriamycin in
0.1 ml of methanol and 10 ml of 0.1 mol/l sodium hydrogenphosphate.
After 2 h, the resulting solution was applied on a column with
Sephadex G-26 and the separated high-molecular-mass fraction was
lyophilized. The resulting preparation contained adriamycin bonded
to 9.6% of monomer units.
EXAMPLE 12
50 mg of the polymer prepared in example 2 was disolved in 10 ml of
0.1 mol/l hydrochloric acid and cooled to 0.degree. C. Under
stirring and cooling 10 ml of 0.1 mol/l sodium nitrite (NaNO.sub.2)
was added to it followed, after 5 min by a solution of 40 mg
cytosine in 10 ml of 0.1 mol/l sodium hydrogencarbonate
(NaHCO.sub.3). After 2 h, the high-molecular-mass fraction was
separated by gel filtration through Sephadex G-26. The resulting
preparation contained cytosine bonded to 0.5% of monomer units.
EXAMPLE 13
The reaction was carried out in the same way as in example 11, with
the distinction that 38 mg of cytosine-.beta.-D-arabinofuranoside
hydrochloride (ara-C) was used. The resulting preparation contained
ara-C bonded to 0.6% monomer units.
EXAMPLE 14
The reaction was carried out in the same way as in example 11, with
the distinction that 40 mg of 5-aza-2'-deoxycytidine (Aza-C) was
used instead of adriamycin. The resulting preparation contained
Aza-C bonded to 0.6% of monomer units.
EXAMPLE 15
The reaction was carried out in the same way as in example 9, with
the distinction that a solution containing 30 mg of
4-amino-N-(2-aminoethyl)benzamide hydrochloride (AABH) was used
instead of N-(6-aminohexyl)-2,4-dinitroanilide. The resulting
polymer contained 4-amino-N-(2-aminoethyl)benzamide hydrochloride
(AABH) bonded to 11% of monomer units.
EXAMPLE 16
50 mg of the polymer prepared in example 15 was dissolved in 2.5 ml
of 0.5 mol/l hydrochloric acid (HCl) and cooled to 0.degree. C.
Under stirring and cooling 0.25 ml of 1 mol/l sodium nitrite
(NaNO.sub.2) was added to it followed, after 5 min by 1 ml of 2
mol/l sodium carbonate (Na.sub.2 CO.sub.3) and 0.26 ml of 1 mol/l
sodium hydrogensulfite, and, after another 5 min, 9 mg of
pancreatic trypsin and kallikrein inhibitor (TKI, specific
antitryptic activity 4.2 BAPA U/mg) in 1 ml water. The non-bonded
trypsin and kallikrein (TKI) were separated from the
high-molecular-mass fraction by gel filtration through Sephadex
G-50. The resulting high-molecular-mass preparation contained 17
weight-% trypsin and kallikrein inhibitor (TKI) and exhibited 0.15
antitryptic BAPA U/mg.
EXAMPLE 17
Adriamycin (10 mg) in 0.4 ml of methanol was added to the solution
of 45 mg of the polymer prepared in example 1 in 10 ml of 0.1 mol/l
acetate buffer with pH 5.05. After 7 days, the high-molecular-mass
fraction was separated by gel filtration through Sephadex G-25 and
freeze-dried. The resulting preparation contained adriamycin bonded
to 11% of monomer units.
* * * * *